38 PRODUCTION OF TRANSGENIC DOGS THAT OVEREXPRESS PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-ALPHA IN A MUSCLE-SPECIFIC MANNER

Abstract

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Among 3 major types of PPAR (PPARα, PPARβ, and PPARγ), PPARα is expressed in tissues having a high rate of β-oxidation, such as brown adipose tissue, liver, kidney, heart, and skeletal muscle. The purpose of this study was to produce transgenic dogs that overexpress PPARα in a muscle-specific manner. Male beagle fetal fibroblasts were infected with viruses containing PPARα under a human myoglobin promoter and green fluorescent protein (GFP) under an EF1 promoter. Stable infectants were isolated with puromycin, and stored in liquid nitrogen until somatic cell nuclear transfer. Natural oestrus and ovulation of 22 dogs (13 oocyte donors and 9 recipients) was monitored by serum progesterone concentration, and in vivo-matured oocytes were recovered by surgical oviduct flushing. After cumulus cells were removed by repeated pipetting, nuclear materials of the denuded oocytes were removed. A GFP-expressing cell was selected under ultraviolet light, and injected into the enucleated oocyte. Then, the oocyte-cell couplets were fused electrically and activated chemically. Immediately after activation, the cloned embryos were transferred into the oviducts of recipient dogs. Pregnancy diagnosis was performed at least 27 days after embryo transfer by ultrasonography, and serum progesterone concentration, rectal temperature, and fetal heartbeat were monitored for safe delivery. As results, 152 in vivo oocytes were recovered from oocyte donor dogs, and 116 cloned embryos were produced with the infected fetal fibroblast cells. Among them, 113 cloned embryos were transferred into 9 recipient dogs, and 3 were diagnosed as pregnant. Three healthy transgenic cloned puppies were produced, and they could serve as a canine model having enhanced physical performance and obesity resistance. This study was supported by NRF (#2014R1A1A2059928), RDA (#PJ010928032016), IPET (#311062-04-3SB010), Nature Cell, Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.